Hepatic ischemia reperfusion injury (IRI) occurs in all transplanted livers, in trauma, shock, and in elective surgery where blood supply to the liver is temporary interrupted. IRI causes up to 10% of early transplant failures and can lead to significantly higher incidence of acute and chronic rejections. Thousands of patients die every year while waiting for a donor organ. This gloomy scenario, together with the significant prevalence of obesity in the population, has led to an increased need of using suboptimal steatotic livers in transplantation at elevated risks of dysfunction based on their high susceptibility to IRI. In the previous funding period, our results demonstrated that specific matrix metalloproteinase-9 (MMP-9) inhibition profoundly ameliorates IRI in normal livers, and unveiled potential key roles for MMP-2 and TIMP-1 in liver injury. This proposal (i) explores the hepatoprotective properties of MMP-9 inhibition in marginal steatotic liver IRI and (ii) dissects the functions of MMP-2 and TIMP-1 in IR-induced damage in both normal and steatotic livers. We expect that the proposed work will provide fundamental insights on the individual functions of key MMPs/TIMPs, leading to the development of novel therapeutic manipulations that can minimize their detrimental effects while maximizing their beneficial functions in normal and in steatotic liver IRI. Well-established models of partial liver IRI in normal and in steatotic mice, and of ex vivo cold steatotic liver ischemia followed by iso-transplantation in rats will be used to address the following aims: (1) To dissect mechanisms by which Matrix Metalloproteinase-9 specific inhibition affects the IRI outcome in marginal steatotic livers. Using MMP-9-/- deficient mice and specific therapeutic manipulations against MMP-9, we will dissect whether specific MMP-9 inhibition (i) protects marginal steatotic livers from the I/R insult, (ii) disrupts leukocyte traffic and chemokine release/activation, and we will (iii) identify intracellular signaling mechanisms leading to MMP-9 expression in steatotic hepatic IRI; (2) To analyze mechanisms by which Matrix Metalloproteinase-2 activity affects the IRI outcome in normal and in steatotic livers. We will determine whether selective MMP-2 inhibition (i) affects liver function/preservation, (ii) results in altered expression of MMP-9, (iii) interferes with patterns of leukocyte migration and of cytokine/chemokine activation and, furthermore, we will (iv) identify the sources of MMP- 2 and their relative contribution in normal and steatotic liver IRI; and (3) To dissect the mechanisms by which tissue inhibitor of metalloproteinases-1 affects IRI in normal and in steatotic livers. We will assess the function of TIMP-1 in normal and in steatotic liver IRI by determining the impact of TIMP-1 deficiency on (i) liver function/preservation, (ii) liver regeneration and apoptosis, (iii) MMP activation, and on (iii) leukocyte recruitment and pro-inflammatory networks. Moreover, we will assess the function of Timp-1 overexpression as a therapeutic approach in partial liver IRI and in steatotic OLT. PUBLIC HEALTH RELEVANCE: Thousands of patients die every year while waiting for a donor organ; this serious situation has led to an increased use of suboptimal steatotic livers in transplantation. However, it is widely accepted that steatotic livers present very high risks of primary non-function, or dysfunction, based on their increased susceptibility to ischemia and reperfusion injury (IRI). We expect that our proposed work will lead to critical insights on the individual functions of key matrix metalloproteinases (MMPs), leading to the development of novel therapeutic manipulations that, by reducing the detrimental effects of MMPs while keeping their beneficial functions in hepatic IRI, will allow the successful utilization of suboptimal donors in transplantation.